Drosophila Cell Lines Research Articles

A Minimal Murine Msx-1 Gene Promoter: ORGANIZATION OF ITS cis-REGULATORY MOTIFS AND THEIR ROLE IN TRANSCRIPTIONAL ACTIVATION IN CELLS IN CULTURE AND IN TRANSGENIC MICE

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing -165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly expressed beta-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1 promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice.

Functional equivalence of native light-sensitive channels in the Drosophila trp 301 mutant and TRPL cation channels expressed in a stably transfected Drosophila cell line

Drosophila photoreceptors express two putative cation channels encoded by the transient receptor potential ( trp) and trp-like ( trpl) genes, which represent prototypical members of a novel family of phosphoinositide-regulated calcium influx channels. Mutations of both trp and trpl selectively abolish components of the light-sensitive current and, when heterologously expressed, both generate cation permeable conductances; however, a detailed comparison of recombinant and native channel properties is lacking. To more rigorously test the hypothesis that TRPL channels mediate one component of the light-sensitive current we have generated cell lines ( Drosophila S2 cells) stably transfected with trpl cDNA and compared the recombinant channel properties with those of the light-sensitive conductance in situ in a Drosophila trp mutant under identical conditions. We found close correspondence in respect of a number of quantifiable biophysical parameters including: current voltage relationships, ionic selectivity, voltage independent block by external Mgt+ ions and effective single channel conductance and gating kinetics derived by noise analysis. Our estimate of 60–70 pS for channel conductance was confirmed directly in patch clamp recordings of single TRPL channels in S2 cells. These findings indicate that channels encoded by the trpl gene can completely account for the component of the light-sensitive conductance remaining in the trp mutant.

Temperature-sensitive expression of Drosophila neuronal nicotinic acetylcholine receptors.

Heterologous expression of cloned Drosophila nicotinic acetylcholine receptor (nAChR) subunits indicates that these proteins misfold when expressed in mammalian cell lines at 37 degrees C. This misfolding can, however, be overcome either by growing transfected mammalian cells at lower temperatures or by the expression of Drosophila nAChR subunits in a Drosophila cell line. Whereas the Drosophila nAChR beta subunit (SBD) cDNA, reported previously, lacked part of the SBD coding sequence, here we report the construction and expression of a full-length SBD cDNA. We have examined whether problems in expressing functional Drosophila nAChRs in either Xenopus oocytes or mammalian cell lines can be attributed to an inability of these expression systems to assemble correctly Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, we have been unable to detect functional nAChRs in a Drosophila cell line unless Drosophila nAChR subunit cDNAs are coexpressed with vertebrate nAChR subunits. Our results indicate that the folding of Drosophila nAChR subunits is temperature-sensitive and strongly suggest that the inability of these Drosophila nAChR subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for coassembly with as yet unidentified Drosophila nAChR subunits.

Baculovirus immediate-early promoter-mediated expression of the Zeocin ™ resistance gene for use as a dominant selectable marker in Dipteran and Lepidopteran insect cell lines

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 μg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 μg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus ( OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus ( AcMNPV) were used to drive expression of the Zeocin resistance gene ( ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3–4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by β-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.

Constructing artificial antigen-presenting cells from Drosophila cells.

Stimulation of unprimed T cells is controlled by professional antigen-presenting cells (APC) such as dendritic cells (DC).1–3 The strong APC function of DC is presumed to reflect that these cells express a high density of major histocompatibility complex (MHC) molecules and a variety of costimulatory molecules. In this respect, activation of naive T cells by APC is thought to require two distinct signals: Signal t reflects T cell receptor (TCR) contact with specific peptides bound to MHC molecules, and Signal 2 is the consequence of other molecules on T cells, e.g. CD28, interacting with costimulatory molecules, e.g. B7 (B7-1, B7-2), on APC. Since many different accessory molecules on APC can express costimulatory function for T cells under defined conditions, which particular accessory molecules are essential for stimulating naive T cells is unclear. We have addressed this issue by constructing artificial APC from a Drosophila cell line by gene transfection.KeywordsMajor Histocompatibility ComplexMajor Histocompatibility Complex ClassProliferative ResponseCostimulatory MoleculeDrosophila CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

UVB Irradiation Upregulation of the Drosophila 1731 Retrotransposon LTR Requires the Same Short Sequence of U3 Region in a Human Epithelial Cell Line as in Drosophila Cells

Phylogenetic analysis of the retrotransposon and retrovirus suggests an evolutionary relationship between them and indicates that transactivation of the long terminal repeat (LTR)-containing retroelements could be ubiquitous. Using constructs expressing a reporter gene under the control of the entire or deleted LTR of 1731, which is a retrotransposable element of Drosophila melanogaster, we were able to show that the UVB-irradiation activation of the 1731-LTR requires the same short sequence of U3 region in a human epithelial cell line as in Schneider's Drosophila cell line (S2). This sequence is similar to the binding sequence of the members of the nuclear factor-kappa B (NF-kappa B)/rel family. In addition, human colonic carcinoma cells (HT29), in response to UVB-irradiation, produce some extracellular factor(s) that activates the 1731-LTR in nonirradiated cells.

Translational Regulationin Vivoof the Drosophila Melanogaster mRNA Encoding Succinate Dehydrogenase Iron Protein via Iron Responsive Elements

Some mRNAs encoding proteins related to iron metabolism contain a specific stem-loop structure—iron responsive element (IRE)-in the 5′ UTR. Binding of the iron regulatory protein (IRP) to the IRE, in response to decreases in cellular iron levels, leads to a block in translation of these mRNAs. We here describe the drosophila melanogaster succinate dehydrogenase iron protein (SDH-IP) as a fourth example of an mRNA species being translationally regulated by an IRE, based on iron dependent regulation of SDH-IP translationin vivoby immunoprecipitations and northern blotting in drosophila cell lines. Addition of hemin to the drosophila cells lead to fragmentation of the SDH-IP, which might suggest an additional mode to specifically down-regulate the expression of this protein and Krebs cycle function.

HLH106, a Drosophila transcription factor with similarity to the vertebrate sterol responsive element binding protein.

We cloned a Drosophila homolog to the sterol responsive element binding proteins (SREBPs). In vertebrates, the SREBPs are regulated by a mechanism that involves cleavage of the protein that normally residues in the cellular membranes and translocation of the released transcription factor into the nucleus. Regulation of the Drosophila factor HLH106 apparently follows the same mechanism, and we find the full-length gene product in the membrane fraction and a shorter cross-reacting form in the nuclear fraction. This nuclear form, which may correspond to proteolytically activated HLH106, is abundant in the blood cell line mbn-2. The general domain structure of HLH106 is very similar to that in SREBP. HLH106 is expressed throughout development, and it is present at high levels in Drosophila cell lines. In contrast to the rat homolog, HLH106 transcripts are not more abundant in adipose tissue than in other tissues.

Wild-type and Insecticide-resistant Homo-oligomeric GABA Receptors of Drosophila melanogaster Stably Expressed in a Drosophila Cell Line

RDL is an ionotropic GABA receptor subunit, a product of the Rdl gene, originally identified in the Maryland strain of Drosophila melanogaster. Here, we report the generation of a Drosophila melanogaster cell line (S2-RDLA302S) stably expressing a mutated, dieldrin-resistant (A302S) form of RDL. The properties of this dieldrin-resistant, homo-oligomeric receptor have been compared with those of the stably expressed, wildtype form (S2-RDL). Using these stable lines, a striking reduction in sensitivity to both picrotoxinin and dieldrin was observed for responses to GABA of S2-RDLA302S compared to S2-RDL. To determine if these stable insect cell lines generate results similar to those obtained by transient expression in Xenopus laevis oocytes, we have examined the actions of two widely used convulsants, EBOB and TBPS, and a recently developed convulsant BIDN, on RDL-mediated GABA responses in the two expression systems. In both oocytes and S2 cells, the three convulsants suppressed the amplitude of responses to GABA. Thus, in accord with earlier work on agonist and allosteric sites, the S2-RDL cell line is found to yield similar pharmacological results to those obtained in transient expression studies. Stable cell lines are now available expressing susceptible and resistant forms of an ionotropic receptor targetted by GABAergic insecticides. Copyright © 1996 Elsevier Science Ltd.

1235 A protein kinase inhibitor, h-7, induces apoptosis in a drosophila neuronal cell line

To investigate the role of microfilaments in natural killer (NK) cell-mediated cytotoxicity, general microfilament inhibitors, cytochalasins B,D and dihydrocytochalasin B, and a selective inhibitor of myosin light chain kinase (MLCK) which regulates microfilament contraction, i.e. 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9) were examined in an NK assay system. ML-9 inhibited NK cell activity in a dose-dependent manner without affecting target cell binding, whereas cytochalasins suppressed the binding. The dextran suspension method revealed that ML-9 inhibits the programming for the lysis stage of the lytic process. In the single cell assay, the addition of ML-9 after target cell binding had occurred inhibited the lysis of bound target cells, whereas the addition of cytochalasins in a similar manner did not affect it. Thus, these results suggest the possibility that microfilament contraction is involved in the lytic mechanism of NK cell-mediated cytolysis. However, the mechanism whereby cytochalasins inhibit target cell binding remains unclear.

Functional Expression of a Cloned Drosophila Muscarinic Acetylcholine Receptor in a Stable Drosophila Cell Line

A cloned Drosophila muscarinic acetylcholine receptor (mAChR) has been stably expressed in a Drosophila cell line (S2) under the control of an inducible Drosophila metallothionein promoter. A clonal cell line (S2-Dm1-1) has been isolated which, after induction of mAChR expression with CuSO4, exhibits high-affinity, saturable, specific binding of the muscarinic antagonist N-methyl scopolamine (NMS). The apparent molecular mass of the expressed protein, calculated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), is in good agreement with the apparent molecular mass of mAChRs purified from Drosophila brain. Functional expression of the cloned mAChR in this stable cell line has been demonstrated by quantitative fluorescence ratio-imaging of Fura-2-loaded cells. We have observed transient, agonist-induced elevations in intracellular Ca2+ levels which can be completely blocked by atropine, whereas AFDX-116, a muscarinic antagonist which binds preferentially to the vertebrate mAChR M2 subtype, has little effect at 100 mumol l-1. The suitability of this stable Drosophila expression system for the characterization of neurotransmitter receptors is discussed.

Calcium Destabilizes Drosophila Cactus Protein and Dephosphorylates the Dorsal Transcription Factor

The Drosophila cactus and dorsal proteins are required for the development of embryonic dorso-ventral polarity and probably also for the innate immune response of the insect. Like their mammalian counterparts (the cytoplasmic anchor protein IκB and the rel/NFκB transcription factors) cactus and dorsal are regulated at the level of nuclear localisation. We showed previously that increased intra-cellular calcium levels induced by the ionophore ionomycin can activate dorsal/cactus complexes in the Drosophila cell line SL2. In order to study further the activation of dorsal/cactus complexes by calcium, we have prepared a cell line (SLDL) in which dorsal is expressed constitutively. In this paper we show that in SLDL cells ionomycin induces a rapid destruction of cactus and dephosphorylation of dorsal. These results suggest a role for the protein phosphatase calcineurin in calcium mediated activation of dorsal/cactus complexes. They also indicate that in the resting cell constitutive phosphorylation of dorsal is in equilibrium with calcium dependent dephosphorylation.

5314813 Drosophila cell lines expressing genes encoding MHC class I antigens and capable of assembling empty complexes and methods of making said cell lines

5314813 Drosophila cell lines expressing genes encoding MHC class I antigens and capable of assembling empty complexes and methods of making said cell lines

Heavy Chain Dimers as Well as Complete Antibodies Are Efficiently Formed and Secreted from Drosophila via a BiP-mediated Pathway

We have constructed a stable Drosophila cell line co-expressing heavy chain (HC) and light chain (LC) immunoglobulins of a humanized monoclonal antibody (mAb) that recognizes the F antigen of respiratory syncytial virus (Tempest, P. R., Bremmer, P., Lambert, M., Taylor, G., Furze, J. M., Carr, F. J., and Harris, W. J. (1991) Bio/Technology 9, 266-271. These cells efficiently secrete antibody with substrate binding activity indistinguishable from that produced from vertebrate cell lines. Significantly, the Drosophila homologue of the immunoglobulin binding chaperone protein (BiP), hsc72, was found to interact specifically with the immunoglobulin HC in an ATP-dependent fashion, similar to the BiP-HC interaction known to occur in vertebrate cells. This is, in fact, the first substrate ever shown to interact specifically with Drosophila hsc72. Most surprisingly, expression of heavy chains in the absence of LC led to the efficient secretion of heavy chain dimers. Moreover, this secretion occurred in association with hsc72. This dramatically contrasts with what is seen in vertebrate cells where in the absence of LC, HC remains sequestered inside the cell in stable association with BiP. Our results clearly suggest that Drosophila BiP can substitute for its mammalian counterpart and chaperone the secretion of active IgG. However, the finding that Drosophila BiP can also uniquely chaperone heavy chain dimers indicates mechanistic differences that may relate to the evolved need for retaining immature IgGs in vertebrates.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is targeted to the cytoplasm in insect cell lines

Cytosolic NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase synthetase and the mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) are differentially expressed during insect development although both enzymes are detectable at all stages. In contrast, cell lines derived from a variety of insect species express high levels of NMDMC but undetectable levels of the NADP-dependent enzyme. Northern analysis indicates the NMDMC message is expressed at levels 50–100 times higher in a Drosophila cell line compared to adult flies. RNase protection showed the predominance of shortened transcripts that require initiation at a downstream AUG producing a truncated protein that lacks a mitochondrial targeting sequence. These changes in expression effectively exchange the cytosolic NADP-dependent dehydrogenase for one with NAD specificity.

5314813 Drosophila cell lines expressing genes encoding MHC class I antigens and B2-microglobulin and capable of assembling empty complexes and methods of making said cell lines

5314813 Drosophila cell lines expressing genes encoding MHC class I antigens and B2-microglobulin and capable of assembling empty complexes and methods of making said cell lines

Stable expression of a functional homo-oligomeric Drosophila GABA receptor in a Drosophila cell line

A cloned Drosophila gamma-aminobutyric acid GABA receptor subunit (Rdl) has been stably expressed as a functional homo-oligomeric ion channel in a Drosophila cell line. Stably-transfected clonal cell lines which expressed high levels of GABA receptor were identified by specific [3H]-muscimol binding. Expression of functional GABA-gated ion channels in these cell lines was demonstrated by electrophysiological recording. Rapid and pronounced rundown of responses to GABA during whole-cell patch clamp recordings was overcome by the inclusion of EGTA in the pipette solution, indicating a possible role for calcium-dependent processes in the functional regulation of this GABA receptor. Relative agonist potencies of the expressed receptor were found to be in the order GABA = TACA > CACA. We have observed a reversible block of the receptor by the convulsant antagonists, picrotoxinin and EBOB, and by the insecticide fipronil. Potentiation of GABA responses was seen with the anaesthetic steroid 5 alpha-pregnan-3 alpha-ol-20-one. No significant effects (either agonist, antagonist or modulatory) were observed with bicuculline (a vertebrate GABAAR antagonist), benzodiazepines or barbiturates (vertebrate GABAAR modulators), or with glycine agonist of the closely related vertebrate glycine receptors). The suitability of this Drosophila stable expression system for the characterization of receptors and ion channels is discussed.

Identification of a Strong Transcriptional Activator for the Copia Retrotransposon Responsible for Its Differential Expression in Drosophila hydei and Melanogaster Cell Lines

We have characterized the regulatory properties of a 72bp sequence located in the 5′ untranslated domain of the Drosophila copia retrotransposon, 3′ to the left LTR, by transient transfection assays with cell lines derived from either Drosophila hydei (DH33 cells) or Drosophila melanogaster (Schneider II and Kc cells). Reporter plasmids were constructed which contained the lacZ gene under the control of either the entire copia LTR with 5′ untranslated domain, or a minimal heterologous promoter flanked with the identified copia regulatory sequences. Upon transfection into the copia-free DH33 cells, the presence of the 72bp sequence resulted for all reporter plasmids in a 100-700 fold increase in expression level -as well as in reporter gene RNA levels- whereas this sequence had no enhancing effect upon transfection of the same plasmids into the copia-containing Schneider II or Kc cells. Moreover, mobility shift assays with the 72bp enhancer sequence disclosed two specific bands of retarded mobility with whole-cell extracts from DH33 cells, whereas no retarded band could be detected, under identical conditions, with extracts from Schneider II cells. UV crosslinking experiments between the enhancer sequence and DH33 extracts revealed a single protein species -of app. mel. wt. 50kD- for both retarded bands, thus strongly suggesting that they simply correspond to the sequential binding of two identical factor molecules to the enhancer sequence. These data demonstrate that the copia-free D. hydei cells express a strong transcriptional activator for the copia element and possible interpretations for the absence of this factor in the copia-containing D. melanogaster cells are discussed in terms of a possible "adaptation" of the "host" (D. melanogaster) to an otherwise highly mutagenic "parasite" (copia with its transcription factor).

Chapter 9 Transformation Techniques for Drosophila Cell Lines

Publisher Summary This chapter focuses on techniques for transforming Drosophila cell lines. The chapter shows how individual lines differ substantially in their transfectabilities by hydroxyapatite-DNA. They may also differ in their precise medium requirements, in their susceptibilities to the toxins used for selection, in superficial and changeable properties like surface adherence, and in the precise catalog of expressed genes. Drosophila cell lines can be maintained in a variety of media: Schneider's medium, D-22, and M3, all of which are now commercially available. The chapter emphasizes on the use of M3 because most cell lines adapt readily to growth in M3 supplemented with 5–10% fetal calf serum (FCS). Some lines require additional supplementation with a mixture of bactopeptone (BP) and extra yeast extract (YE). Because there is substantial clone-to-clone variation in the expression of a transformed plasmid, and because bulk selection of a transfected population generally leads to the retention of only the fastest-growing transformed clone within the starting population, it is frequently useful to clone cells just after transfection.

Relocalization of Drosophila dorsal protein can be induced by a rise in cytoplasmic calcium concentration and the expression of constitutively active but not wild-type Toll receptors.

The generation of dorso-ventral polarity in Drosophila relies on the formation of a nuclear gradient of the rel/nuclear factor kappa B transcription factor dorsal in the pre-cellular syncitial embryo by a process of differential nuclear localization. It is thought that the gradient is formed by activation at ventral positions of the membrane receptor Toll that in turn causes the local dissociation of dorsal from the cytoplasmic anchor protein cactus. Although Toll is related in its cytoplasmic domain to the interleukin-1 receptor little is known about the signal transduction pathways that lead from Toll to the relocalization of dorsal. In this paper we have used immunofluorescence microscopy as a direct assay of dorsal protein nuclear localization in the Drosophila cell line Schneider 2. We find that increased cytoplasmic calcium concentration and the expression of constitutively active Toll receptors can induce the relocalization of dorsal. By contrast, we find that activation of endogenous protein kinase A and expression of wild-type Toll receptors, which activate zen-chloramphenicol acetyltransferase reporter genes in this system, have only a marginal effect on the cellular distribution of the dorsal protein. Treatment of cells with activators of protein kinase C and radical oxygen intermediates, both of which activate nuclear factor kappa B, also has little effect on dorsal protein localization. We propose that different threshold levels of dorsal activation can be established by distinctly regulated signal transduction pathways.